B cell proliferation kit (R&D system, Abingdon, UK). BD CellFix (BD Biosciences, Oxford, UK). Native human FV, FVa, Thrombin, Dynabeads Human T-cell activator, and CFSE kits (Thermofisher Scientific, Loughborough, UK). Human Factor V ELISA Kit (ab137976, Abcam, Cambridge, UK). Unless otherwise indicated, all reagents were from Sigma-Aldrich Company Ltd (Dorset, UK).
CD4 T cell, CD8 T cell and B cell were isolated using Miltenyi isolation kits (Miltenyi Biotec, Surrey, UK) following the manufacturer’s instruction. Cells were stained with CFSE using CFSE kits (Thermofisher Scientific, Loughborough, UK) following the manufacturer’s instruction. 5 × 104 labelled T cons were mixed with Dynabeads Human T cell activator CD3/CD28 (Thermofisher Scientific, Loughborough, UK) at cell-to-beads ratio of 2 to 1 and incubated at 37°C. Cells incubated with native human FV, FVa, FV constructs at the concentrations indicated, thrombin (5 μg/mL), and hirudin (5 iU/ml) were collected and analysed using a BD Fortessa flow cytometer after 4 or 5 days.
CD4 T cell, CD8 T cell and B cell were isolated using Miltenyi isolation kits (Miltenyi Biotec, Surrey, UK) following the manufacturer’s instruction. Cells were stained with CFSE using CFSE kits (Thermofisher Scientific, Loughborough, UK) following the manufacturer’s instruction. 5 × 104 labelled T cons were mixed with Dynabeads Human T cell activator CD3/CD28 (Thermofisher Scientific, Loughborough, UK) at cell-to-beads ratio of 2 to 1 and incubated at 37°C. Cells incubated with native human FV, FVa, FV constructs at the concentrations indicated, thrombin (5 μg/mL), and hirudin (5 iU/ml) were collected and analysed using a BD Fortessa flow cytometer after 4 or 5 days.